The overall goal of this project is to maximize the potential of retrovirus vectors for gene transfer into the airway epithelium of cystic fibrosis (CF) patients. We will focus on the following aspects of gene transfer: (i) improving the efficiency of retrovirus entry into primary human epithelial cells; (ii) optimizing the persistence of transduced cystic fibrosis transmembrane conductance regulator (CFTR) gene expression in human airway epithelium; and (iii) developing methods to maximize gene transfer in vivo. The focus on retrovirus entry into airway epithelial cells involves (a) adaptation of retroviruses to replicate more efficiently these cells; and (b) localizing the GALV receptor in the airway epithelium and to determine how its expression changes during proliferative repair. The focus on transduced CFTR expression involves optimizing long-term expression in CF cells grown in culture and optimizing the persistence of expression during differentiation in vivo using the tracheal graft model. The emphasis on long-term expression is organized around the following functional themes: (a) the role of negative regulatory elements on long term expression; (b) the effect of CFTR over-expression on long term expression; (c) long-term expression using housekeeping promoters; and (d) testing a recently developed splicing vector. The focus on maximizing gene expression in vivo will be to: (a) maximize airway epithelium proliferation rates by exposure to inhaled oxidants coupled with exposure to mitogens; and (b) increase the duration of exposure to retrovirus vectors by the delivery of retrovirus packaging cell lines on micro-carrier beads. Our hypothesis is that retroviruses will be useful vehicles for gene transfer into the airways of newborn animals, where the proliferation rate is higher than adults, or alternatively, into the airways of adults whose epithelium is proliferating in response to limited injury, for example after exposure to oxidants. As a critical test of this hypothesis we will determine the efficiency and safety of retrovirus gene transfer to the airways using the rhesus monkey as a non-human primate model.